RESUMEN
The aleurona cell is a model that allows the study of the antagonistic effect of gibberellic acid (GA) and abscisic acid (ABA). Previous results of our laboratory demonstrated the involvement of phospholipids during the response to ABA and GA. ABA modulates the levels of diacylglycerol, phosphatidic acid and diacylglycerol pyrophosphate (DAG, PA, DGPP) through the activities of phosphatidate phosphatases, phospholipase D, diacylglycerol kinase and phosphatidate kinase (PAP, PLD, DGK and PAK). PA and DGPP are key phospholipids in the response to ABA, since both are capable of modifying the hydrolitic activity of the aleurona. Nevertheless, little is known about the mechanism of action of these phospholipids during the ABA signal. DGPP is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol. The ionization of the pyrophosphate group may be important to allow electrostatic interactions between DGPP and proteins. To understand how DGPP mediates cell functions in barley aleurone, we used a DGPP affinity membrane assay to isolate DGPP-binding proteins from Hordeum vulgare, followed by mass spectrometric sequencing. A cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was identified for being bound to DGPP. To validate our method, the relatively abundant GAPDH was characterized with respect to its lipid-binding properties, by fat western blot. GAPDH antibody interacts with proteins that only bind to DGPP and PA. We also observed that ABA treatment increased GAPDH abundance and enzyme activity. The presence of phospholipids during GAPDH reaction modulated the GAPDH activity in ABA treated aleurone. These data suggest that DGPP binds to GAPDH and this DGPP and GAPDH interaction provides new evidences in the study of DGPP-mediated ABA responses in barley aleurone.
Asunto(s)
Difosfatos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glicerol/análogos & derivados , Hordeum/metabolismo , Células Vegetales/metabolismo , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Difosfatos/metabolismo , Glicerol/metabolismo , Glicerol/farmacología , Hordeum/citología , Unión ProteicaRESUMEN
The patch-clamp technique was designed to measure any electrogenic transport across the whole cell and organelle (vacuolar) membranes and excised membrane patches. Here, we describe preparation of protoplasts and vacuoles, as well as patch-clamp assays, to detect the functional expression of K(+) and cation channels of plasma membrane and tonoplast, as well as plasma membrane anion channels and vacuolar and plasma membrane H(+) pumps. All of these contribute to the intracellular ionic homeostasis under saline conditions.
Asunto(s)
Transporte Iónico , Proteínas de Transporte de Membrana/metabolismo , Técnicas de Placa-Clamp/métodos , Células Vegetales/metabolismo , Cloruro de Sodio , Homeostasis , Hordeum/citología , Hordeum/metabolismo , Canales Iónicos/metabolismo , Iones/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Bombas de Protones/metabolismo , Protoplastos/citología , Protoplastos/metabolismo , Semillas/crecimiento & desarrollo , Soluciones , Vacuolas/metabolismoRESUMEN
The barley plastome mutant CL2 (cytoplasmic line 2) carries a point mutation in the infA gene, a homologue of the bacterial gene for the conserved translation initiator factor 1 (IF1). The function of infA in plastids is not known. The mutation in CL2 leads to a temporal chlorophyll deficiency in the primary leaf blade that is normalised in the basal and middle parts during further development. We have compared the expression of selected nuclear and plastid genes in different parts of primary leaves of CL2 and wild-type and found no indication for an adverse effect of the mutation on plastidial transcription. We observed an enhanced expression of RpoTp (encoding the phage-type nuclear-encoded plastid RNA polymerase) suggested to be caused by retrograde plastid signalling. Decreased amounts of plastid rRNA in basal and top sections are in agreement with the idea that the mutation in infA leads to a time- and position-dependent defect of plastid translation that causes a delay in plastid development. The normalisation of the phenotype in the middle section of CL2 leaves correlates with wild-type levels of chloroplast 16S rRNA and RbcL and increased expression of plastid housekeeping genes. The normalisation was not observed in cells at the tip of CL2 leaves suggesting different ways of regulating chloroplast development in cells at the tip of primary barley leaves as compared with other leaf sections.
Asunto(s)
Genoma del Cloroplasto , Hordeum/genética , Secuencia de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Senescencia Celular/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Cartilla de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Genes de Plantas , Hordeum/citología , Hordeum/metabolismo , Microscopía Electrónica de Transmisión , Modelos Biológicos , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Mutación Puntual , Transcripción GenéticaRESUMEN
Hordeum chilense Roem. et Schult. has a number of characteristics interesting for breeding: high crossability with other Triticeae, resistance to biotic and abiotic stresses and high variability for quality traits such as endosperm storage proteins or carotenoid content. xTritordeum, the amphiploids between H. chilense and different Triticum spp, are bridge species which facilitate the transfer of traits from H. chilense to wheat or triticale. The chromosome pairing between H. chilense and wheat chromosomes is very low (if existing) even in the absence of the action of the Ph1 gene. Nevertheless, translocation between H. chilense and wheat chromosomes has been observed frequently in genomic combinations where univalents of both species are present and therefore a method is available for using H. chilense in wheat or triticale breeding. Hybrids and amphiploids with other crop species of the Triticeae, such as rye or barley, have also been obtained, although to date the production of stable introgression stocks has not been completed. The technique of chromosome painting, using both high- and low-repeated DNA sequences in combination with genomic in situ hybridization have been used as effective methods for basic cytogenetic research in H. chilense, allowing analysis of genome evolution, and monitoring H. chilense chromosomes in interspecific hybridization breeding programs.
Asunto(s)
Hordeum/citología , Hordeum/genética , Cruzamiento/métodos , Mapeo Cromosómico , Emparejamiento Cromosómico/genética , Cromosomas de las Plantas/genética , ADN de Plantas/genética , ADN Ribosómico/genética , Genoma de Planta , Secale/genética , Translocación Genética , Triticum/genéticaRESUMEN
Barley (Hordeum vulgare L.) and wheat (Triticum monococcum L. and Triticum turgidum L.) were among the principal 'founder crops' of southwest Asian agriculture. Two issues that were central to the cultural transition from foraging to food production are poorly understood. They are the dates at which human groups began to routinely exploit wild varieties of wheat and barley, and when foragers first utilized technologies to pound and grind the hard, fibrous seeds of these and other plants to turn them into easily digestible foodstuffs. Here we report the earliest direct evidence for human processing of grass seeds, including barley and possibly wheat, in the form of starch grains recovered from a ground stone artefact from the Upper Palaeolithic site of Ohalo II in Israel. Associated evidence for an oven-like hearth was also found at this site, suggesting that dough made from grain flour was baked. Our data indicate that routine processing of a selected group of wild cereals, combined with effective methods of cooking ground seeds, were practiced at least 12,000 years before their domestication in southwest Asia.